1. Field of Invention
The present invention relates to a simple, efficient, safe, economical, fast and improved transmembrane loading procedure for efficient loading of amphiphatic drugs and chemicals into liposomes using the transmembrane gradient. The resulting liposomes loaded with the amphiphatic drug are stable and safe. The procedure is equally applicable for sustained release of liposome encapsulated drugs.
2. Related Disclosures
In recent years the pharmaceutical sciences discovered liposomes, lipid based carrier vehicles as a new formulating entity. Currently, both conventional or nonphospholipid liposomes are rapidly becoming accepted as pharmaceutical agents which improve the therapeutic activity of a wide variety of compounds and provide a convenient drug delivery system. Liposome drug delivery systems are reviewed in detail in Cancer Res., 43:4730 (1983), Pharmacol. Rev. 36:277-336 (1984), Liposomes as Drug Carriers, Gregoriadis, G. Ed., Wiley, New York (1988).
Liposomes are suitable delivery vehicles for parenteral, peroral, topical and inhalation administration of drugs. Liposomes improve formulability, provide prolonged release, improve the therapeutic ratio, prolong the therapeutic activity after each administration and reduce the need for frequent administration, reduce the amount of drug needed and/or absorbed by the mucosal or other tissue.
Loading of the drugs into liposomes has proved to be a measure of their utility. If there is a poor loading, there is a great loss of the active drug and the use of liposomes as the pharmaceutical vehicle becomes uneconomical. In recent years considerable effort has been dedicated to development of systems for various methods of loading of drugs and biological materials into liposomes. Pharmacol. Rev., 36: 277-336 (1984). Liposomes as Drug Carriers, Gregoriadis, G. ed. Wiley, New York, (1988).
So far, several methods have been developed for liposome drug loading. The simplest method of drug loading is a passive entrapment of water soluble drug in the dry lipid film by hydration of lipid components. The loading efficiency of this method is generally low because it depends on the entrapping volume of the liposomes and on the amount of lipids used to prepare them. Chem. Phys. Lipids, 40:333-345 (1986), and Methods in Biochemical Analysis, 33:337 (1988). Disadvantages of this method are the low entrapment, heterogeneous size and the need for secondary processing steps such as extrusion or sonication. Improved passive entrapment of a drug into liposomes has been achieved by using dehydration-rehydration method where preformed liposomes are added to an aqueous solution of the drug and the mixture is dehydrated either by lyophilization, evaporation, or by freeze-thaw processing method involving repeated freezing and thawing of multilamellar vesicles which improves the hydration and hence increases a loading. Disadvantages of these methods are heterogeneous size, difficult standardization and low reproducibility.
Higher efficiency of drug entrapment into liposomes may be achieved by using high lipid concentration or by specific combination of lipid components. For example, an amphiphatic amine doxorubicin may be encapsulated more efficiently into liposome membranes containing negative charge. Cancer Res., 42: 4734-4739 (1982). However, in general the drug loading remains a problematic issue.
The efficiency of drug loading into liposomes depends also on chemical properties of the drug. In general, water soluble or lipid soluble drugs are easier to deal with since the lipid soluble compounds easily incorporate into the lipid bilayer during the liposome formation and the water soluble compounds interact with the polar head group of phospholipids facing inside of liposome and are therefore sequestered inside the liposomes. Amphiphatic compounds, on the other hand, are the most difficult to retain inside the liposomes as they can rapidly permeate through and do not bind to lipid bilayers.
One proposed method of loading amphiphatic molecules into liposomes, described in Chem. Phys. Lipids, 40:333-345 (1986), is drug loading in response to the ion pH gradient by an accumulation of the amphiphatic drug into liposomes when their internal pH is lower than the external medium pH.
PCT/US 87/01401, filed Jun. 16, 1986 describes asymmetrical liposome vesicles containing ionizable lipids or ionizable proteins made in an aqueous environment of controlled pH, then exposed to a bathing medium of a relatively more acidic or relatively more basic pH.
Loading of amphiphatic drugs into the liposomes, described in the J. Biol. Chem., 260:802-808 (1985), utilizes the transmembrane Na.sup.+ /K.sup.+ gradients. Improved loading of local anesthetic dibucaine was observed only when both sodium and potassium ions were used and up to about 52% loading was achieved when the sodium/potassium gradient was used in combination with valinomycin. Valinomycin is known insecticide, nematocide and bactericide as well as ionophore and is thus not desirable additive to pharmaceutical formulations. The method of passive entrapment of the antineoplastic drugs into the liposomal vesicles, described in Biochim. Biophys. Acta, 816:294-302 (1985) uses the uptake of the drugs into the vesicles in response to a valinomycin-dependent K.sup.+ diffusion potential.
Another method of loading of amphiphatic drug adriamycin into liposomal vesicles, described in Biochim. Biophys. Acta, 857:123-126 (1986), is an uptake of the drug into the liposomes in response to the pH gradient. This method would seem to be a reasonably efficient for loading if not for the fact that it is performed in unphysiologically acidic pH and in the presence of the strong base KOH, both of which cause the lipid hydrolysis. Also, the obtained liposome-drug vesicles are unstable and around 24 hours suffer from a substantial leakage of the drug.
PCT/US85/01501 filed on Aug. 7, 1985, describes encapsulation of antineoplastic agents into liposomes by passive loading, via transmembrane potential, of lipophilic ionizable antineoplastic drugs which can exist in a charged state when dissolved in an aqueous medium. The proton gradient is established by first trapping (passively) a low pH buffer and then raising the external pH by adding the base. The transmembrane potential is achieved by sodium/potassium chemical gradient in the presence of valinomycin. The loading is temperature dependent and the best results are achieved at the temperature of 60.degree. C.
A high encapsulation of method for loading water soluble drugs in liposomes has recently been described in U.S. Pat. No. 4,752,425. However, the disadvantage of this method is that it is based on passive entrapment at high lipid concentration.
There are several major disadvantages connected with the previously known methods. First, there is a prolonged exposure of the liposomal lipids to a low acidic pH environment of about pH 4 which leads to lipid hydrolysis and a drug leakage from liposomes. Second, these methods require very high lipid concentration. Third, long periods of time and elevated temperatures are required for loading which leads to lipid hydrolysis and/or drug deactivation. Finally, there are stability problems connected with these methods.
The current invention provides simple, fast, stable, economical, safe, and efficient system for active liposome drug loading and controlled sustained time release by creating an ammonium (NH.sub.4.sup.+) gradient between two sides of a liposome vesicle membrane. The system may be actively manipulated by diluting, removing or exchanging outside ammonia medium to achieve faster or slower, larger or smaller drug loading. The system is not method dependent, i.e. all methods of liposome preparation and all types of liposomes may be used in practicing this invention. The system is also useful for ultrasound imaging by using ammonium gradient in liposomes as a source of hyperechogenic CO.sub.2 which enhances ultrasound imaging.